Despite the significant clinical consequences of cardiac conduction system (CCS) disease, the molecular mechanisms that control CCS function are unknown. This proposal combines novel innovations with recent progress to experimentally dissect the transcriptional control of ventricular conduction system (VCS) function, a problem of great clinical significance. We have developed a novel VCS-specific inducible Cre transgenic mouse line (minKCreERT2) and utilized it to remove Tbx5 from the mature VCS (Tbx5MinK: CreERT2). The normally fast conducting VCS became functionally slow in Tbx5MinK:CreERT2 mice, with concomitant molecular alterations of VCS gene expression. Importantly, Tbx3, required for slow-conducting nodal phenotype was maintained. These observations and recent literature coalesce in the Overall Hypothesis: A Tbx5 / Tbx3 T-box code determines the functional and molecular regional phenotype of the mature Ventricular Cardiac Conduction System. We predict that Tbx5 determines VCS functional and molecular identity and that Tbx3 is required for underlying nodal potential of the VCS, uncovered in the absence of Tbx5. We propose to (1) Test the hypothesis that Tbx5 promotes functional and molecular VCS phenotype; (2) Test the hypothesis that Tbx5 directly regulates SCN5A expression in the VCS to control a molecular hierarchy required for VCS function; (3) Test the hypothesis that Tbx3 is sufficient for establishing a nodal phenotype in the VCS; and (4) Test the hypothesis that Tbx5 and Tbx3 cooperatively promote specialized CCS phenotype. A group of the leading experts in molecular analysis of the CCS have been assembled to investigate the transcriptional control of mature VCS function. A comprehensive experimental set will establish the specific roles of Tbx5 and Tbx3 in the transcriptional control of CCS regional specificity. These investigations will establish a platform for future efforts to detail transcriptional networks governing CCS function, aiding knowledgeable development of clinical CCS therapies and interventions. PUBLIC HEALTH RELEVANCE: Despite the significant clinical consequences of cardiac conduction system (CCS) disease, the molecular mechanisms that control CCS function are unknown. This proposal combines novel innovations with recent progress to experimentally dissect the transcriptional control of ventricular conduction system (VCS) function, a problem of great clinical significance. These investigations will establish a platform for future efforts to dtail transcriptional networks governing CCS function, aiding knowledgeable development of clinical CCS therapies and interventions.